Tunnels for Protein Export from the Endoplasmic Reticulum.

The functions of coat protein complex II (COPII) coats in cargo packaging and the creation of vesicles at the endoplasmic reticulum are conserved in eukaryotic protein secretion. Standard COPII vesicles, however, cannot handle the secretion of metazoan-specific cargoes such as procollagens, apolipoproteins, and mucins. Metazoans have thus evolved modules centered on proteins like TANGO1 (transport and Golgi organization 1) to engage COPII coats and early secretory pathway membranes to engineer a novel mode of cargo export at the endoplasmic reticulum.

Deceased Donor Renal Transplantation: 6 Year Experience from a Tertiary Care Center in North India.

The incident of End stage renal disease (ESRD) is rising rapidly worldwide. Renal transplant is the best modality of treatment, offering a better quality of life and mortality benefit, as compared with long-term dialysis. Very few patients have a live renal transplant donor, for rest, a decreased donor renal transplant is the only alternative. Deceased donor renal transplantation (DDRT) programs are only available at few government centers of India, constituting less than 5% of the total renal transplants. MATERIAL: The patients who had undergone DDRT at our center from February 2015 to February 2021 were registered in the study. The following data were recorded for all patients; age, sex, duration of ESRD, cold ischemia time, type of induction, nadir and follow -up creatinine, hemoglobin, urinary protein and complications. All recipients were followed up and investigated in the outpatient department on a regular basis as per the standard guidelines till death or graft loss, whichever is earlier. Post transplant renal allograft function was measured using serum creatinine and other parameters. OBSERVATION: During the study period 51 DDRTs were done. There were 40 male and 11 female patients. The mean age was 39.9 ± 9.8 years. The most common cause of ESRD in recipients was chronic glomerulonephritis (CGN) in 92.1 % (47). Amongst the patients, 41 (80.3%) survived, while 10 (19.6%) died post-transplant. Out of ten, 6 recipients died due to early sepsis (<3 months) and 4 died due to late sepsis (>3 months). Acute rejection was present in 17.6 % of patients. Mean post- transplant creatinine in recipients with functioning graft at discharge was 1.54 mg/dl. Graft failure was present in 7 patients out of which 2 were alive at the time of writing this paper and were on maintenance dialysis. Two patients died with a functioning graft. Delayed graft function (DGF) was seen in 13.7% (n=7) of recipients. The causes of DGF in our study included transplant renal artery thrombosis (n=2), Antibody-Mediated Rejection (n=3), mixed rejection (n=1) and Acute cellular rejection (n=1). Among those who had DGF, graft loss was seen in 57.2% (n=4). CONCLUSION: In our study, the patient survival and graft survival have been better as compared to previous studies and also the number of recipients with delayed graft function have been low. Deceased donor renal transplantation is a practical treatment modality which can drastically improve longevity and quality of life.

Lepromatous leprosy: An Unusual Presentation.

A 25 year old man presented with loss of sensations over both hands and feet and extreme difficulty in passing urine. On examination, an indurated sclerotic plaque was present on shaft of penis and scrotum in addition to other features of lepromatous leprosy. Skin biopsy from the penile lesion showed presence of a large number of acid fast bacilli with a BI of 6+ extending into the epidermis and histopathology showed absence of Grenz zone and presence of foamy macrophages in the dermis up to the dermo-epidermal junction.

The prevalence of abnormal ECG in trained sportsmen.

BACKGROUND: Competitive sports training causes structural and conductive system changes manifesting by various electrocardiographic alterations. We undertook this study to assess the prevalence of abnormal ECG in trained Indian athletes and correlate it with the nature of sports training, that is endurance or strength training. METHODS: We evaluated a standard resting, lying 12 lead Electrocardiogram (ECG) in 66 actively training Indian athletes. Standard diagnostic criteria were used to define various morphological ECG abnormalities. RESULTS: 33/66 (50%) of the athletes were undertaking endurance training while the other 33 (50%) were involved in a strength-training regimen. Overall 54/66 (81%) sportsmen had significant ECG changes. 68% of these changes were considered as normal training related features, while the remaining 32% were considered abnormal. There were seven common training related ECG changes-Sinus Bradycardia (21%), Sinus Arrhythmia (16%), 1st degree Atrioventricular Heart Block (6%), Type 1 2nd-degree Atrioventicular Heart Block (3%), Incomplete Right bundle branch block (RBBB) (24%), Early Repolarization (42%), Left Ventricular Hypertrophy (LVH) (14%); while three abnormal ECG changes--T-wave inversion (13%), RBBB(4%), Right ventricular hypertrophy (RVH) with strain (29%) were noted. Early repolarization (commonest change), sinus bradycardia, and incomplete RBBB were the commoner features noticed, with a significantly higher presence in the endurance trained athletes. CONCLUSION: A high proportion of athletes undergoing competitive level sports training are likely to have abnormal ECG recordings. Majority of these are benign, and related to the physiological adaptation to the extreme levels of exertion. These changes are commoner during endurance training (running) than strength training (weightlifting).

Comparative Analysis of the Effects of Exercise and Kapalbhati Pranayama on Heart Rate Variability and Electroencephalogram Activity: Unveiling Physiological and Cognitive Insights.

Physical activity and mindfulness techniques, such as exercise and Kapalbhati Pranayama, are known to have positive effects on health and well-being. However, limited research has directly compared their impact on physiological and psychological parameters. This study aimed to investigate and compare the effects of exercise and Kapalbhati Pranayama on Heart Rate Variability (HRV) and Electroencephalogram (EEG) activity, shedding light on their contributions to overall health and mental well-being. The study was conducted at All India Institute of Medical Sciences (AIIMS) in Bhopal, India, from 2018 to 2022. A prospective interventional design was employed over two months, involving 20 participants with normal BMI and no respiratory or cardiac conditions. Exclusion criteria included lung or cardiac diseases, smoking history, dyspnoea during physical activity, pedal edema, and high blood pressure. The Kapalbhati Pranayama intervention was supervised and limited to 5 minutes. HRV was assessed using the HRV Brain Tap Neuralchek Machine before, during, and after Kapalbhati. For exercise, a mild-intensity cycling protocol was performed on healthy volunteers aged 20 to 50. HRV was recorded before, during, and after exercise. The EEG analysis revealed notable changes in brain wave patterns. At baseline, participants exhibited higher levels of delta, theta, and alpha waves, indicating a state of relaxation and calmness. During exercise, there was a significant increase in beta waves and a decrease in delta, theta, and alpha waves, reflecting heightened brain activity and alertness. After Kapalbhati, beta wave levels remained elevated, while delta and theta wave suppression was more pronounced, suggesting a stimulating effect on the brain similar to exercise. The changes in beta and gamma EEG waves could be attributed to factors such as exercise intensity, duration, frequency, and the release of endorphins during both exercise and Kapalbhati. The HRV analysis demonstrated distinct responses to exercise and Kapalbhati. Exercise led to a significant reduction in HRV parameters, characterized by increased heart rate and decreased time-domain HRV measures, aligning with the typical sympathetic nervous system dominance during physical activity. In contrast, Kapalbhati's impact on HRV parameters was milder, with minor changes in heart rate and subtle alterations in time-domain HRV measures. The high LF/HF ratio during Kapalbhati suggested a potential stimulation of the sympathetic nervous system. More comprehensive research is required to confirm these findings and understand the long-term effects of Kapalbhati on HRV and cardiovascular health. This study contributes to the understanding of how exercise and Kapalbhati Pranayama affect both cognitive and cardiovascular aspects of health. It highlights that both interventions increase brain activity and alertness, but Kapalbhati may have a more potent effect. Exercise significantly reduces HRV parameters, indicating sympathetic nervous system dominance, while Kapalbhati has milder HRV effects. Further research with larger and more diverse populations is essential to confirm and expand on these findings, providing insights into optimizing cognitive function and cardiovascular health through tailored approaches of exercise and Kapalbhati Pranayama.

Vitamin A, C and E levels in patients with Non-Alcoholic Fatty Liver Disease: An Indian Cohort Study.

This study aimed to investigate the levels of vitamins A, C, and E in patients with Non-Alcoholic Fatty Liver Disease (NAFLD) compared to healthy controls and to explore the correlation between these vitamin levels and various other parameters, including bone mineral density (BMD), adiposity (fat storage), insulin resistance and subclinical inflammation. The study involved 50 participants diagnosed with NAFLD and 50 healthy controls. Blood samples were collected to measure vitamin A, C and E levels, along with other parameters like insulin, inflammatory markers, and liver function tests. Additionally, participants underwent DEXA scans to assess BMD and body composition. Vitamin levels: The study found no significant deficiencies in vitamin A or C levels in either group. However, vitamin E levels were significantly higher in the NAFLD group compared to controls, although only one case of vitamin E deficiency was observed in the NAFLD group. No significant correlations were found between vitamin levels and BMD, adiposity parameters, insulin resistance, or subclinical inflammation markers in either group. The study acknowledges the limited data available on the association between NAFLD, vitamin levels and BMD in the Asian Indian population. The findings regarding vitamin A and C levels are consistent with some previous studies, whereas the higher vitamin E levels in the NAFLD group contradict other research. This discrepancy might be due to factors like sample size, dietary habits, or vitamin fortification programs. The lack of significant correlations between vitamin levels and other parameters suggests that further research is needed to understand the complex interplay between these factors in NAFLD development and progression.

Changes in Peripheral Brachial Blood Pressure from Supine to Lateral Decubitus Position in Hypertensive and Normotensive Subjects.

Arterial blood pressure is crucial for supplying the nutritional demands of the all body tissues and is also under tight control for the same. The day to day stress impacts the blood pressure (BP), which is effectively corrected by the baroreceptors mechanism. The effect of lateral decubitus position on the blood pressure with respect to supine position needs to be evaluated as it impacts the haemodynamics. The peripheral brachial blood pressure changes are studied among the normotensives and hypertensives with its significance from change in supine to lateral decubitus position. Sixty (60) normotensives and 60 hypertensive subjects were evaluated for arterial blood pressure measurement using mercury sphygmomanometer by gold standard auscultatory method in the supine, right and left lateral decubitus position. The same observer measured the blood pressure at the similar point of time for all the participants. The Supine and lateral decubitus Systolic and diastolic blood pressure (Mean±SD) parameters were recorded and compared using the paired 't' test (Supine Normotensives SBP 112.5±16.9, DBP 70.1±17.7 mm of Hg; Hypertensives Stage I SBP 130.2±14.5, DBP 85.2±2.8 mm of Hg; Hypertensive Stage II SBP 152.6 ±17.5, DBP 98.4±9.5mm of Hg). The mean and percentage change in systolic and diastolic BP for Normotensives from supine to right lateral position- SBP 11.6 mm of Hg (10.3%), DBP 8.8 mm of Hg (12.5%) and supine to left lateral position SBP 12.5mm of Hg (11.1%), DBP 9.9 mm of Hg (14.1%) p<0.05, for Hypertensives Stage I supine to right lateral position SBP 13.8 mm of Hg (10.5%), DBP 14.8 mm of Hg (17.37%) and supine to left lateral position SBP 13.5 mm of Hg (10.3%), DBP 12.3 mm of Hg (14.13%) p<0.05 and for Hypertensive Stage II from supine to right lateral position SBP 22.6 mm of Hg (14.8%), DBP 16.6 mm of Hg (16.8%), form supine to left lateral position SBP 12.3mm of Hg (8%), DBP 14.7mm of Hg (14.9%) p<0.01) changed significantly among the all the groups with respect to change from supine to lateral decubitus position. The fall in diastolic BP was of greater magnitude than the systolic BP. The right lateral decubitus position was dominant among the hypertensives for producing the change as compared to left lateral decubitus position. However among the normotensives it was left lateral decubitus which produced greater reduction in blood pressure. The lateral decubitus position definitively reduces the blood pressure as compared to supine position and the changes produced can't be simply ascribed to the hydrostatic effect of gravity on blood column. Sleeping on lateral side could thus be advantageous in reducing the arterial blood pressure.

Prevalence and Predictors of Depression, Anxiety and Stress among Elderly during COVID-19: A Cross-sectional Study from Central India.

During the COVID-19 pandemic, elderly people have been more prone to depression, anxiety and stress. During these trying times, they require more attention and support for their mental health. This cross-sectional study was performed with the duration of 06 months from March 2021 to August 2021 in AIIMS, Bhopal in the state of Madhya Pradesh, in central India. And the participants recruited by systematic random sampling from a population aged more than 60 years, those who were able to read and write Hindi or English and having at least one family member; who reported to AIIMS, Bhopal during the second wave of COVID-19 in India. Those who were confirmed COVID-19 cases and undergoing treatment for the same, with diagnosed mental health disorders and who didn't give consent were excluded. A Google form based online semi-structured questionnaire along with DASS-21 scale was completed by participants. Elderly (>=60 years) will be selected. Of the 690 participants 7.25% reported mild to moderate depression, whereas 0.58 percent had severe or extremely severe depression. Mild to moderate anxiety were found in 9.56% of people, while 2.46% had severe or extremely severe anxiety. Mildly or moderately were stressed 4.78%, while 0.42% was severely or extremely anxious. Alcoholism and depression were found to have a statistically significant relationship (p=0.028). During the COVID-19 pandemic, elderly subjects who napped during the day were substantially less depressed (p=0.033). The older the respondents were, the more nervous they were during the pandemic (p=0.042). There is a link between alcohol consumption and stress (p=0.043) and it was seen that females were more stressed as compared to males (p=0.045). There was a strong correlation between participants' alcohol addiction and depressive symptoms. Psychological therapies for the elderly are thought to be necessary to enhance their psychological resilience and mental health. We need to tackle the stigma related to the COVID-19 and mental health issues.

Slow Frequency Anuloma Viloma Pranayama Modulates Cardiac and Neural Oscillations in Yoga Practitioners.

Anuloma viloma pranayama (AVP) also known as Alternate nostril breathing (ANB) is the most commonly used form of pranayama, the ancient breath control practice. It is known to modulate cardiovascular control and brain activity. This study analysed the simultaneous recording of Heart rate variability (HRV) and brain activity during and after AVP at a very slow frequency. We carried out an observational cohort study from October 2021 to December 2021 with purposive sample of 30 (Thirty) healthy volunteers involved in regular kriya yoga practice for the last 15 years were inducted into this study AVP involves slow and deep inhalation through one nostril at a time. The ECG for HRV analysis and spectrum of EEG waves were recorded throughout the pre-AVP, during-AVP and post-AVP (each of five-minute duration). The parameters were compared and analysed by repeated measures of analysis of variance with post-hoc analysis using Bonferroni and Holm's multiple comparisons. In time Domain parameters, SDNN and RMSSD were significantly higher during AVP as compared to the pre-AVP and post AVP. In frequency domain parameters total power, LF power, HF power, showed a significant increase during AVP, LF/HF ratio increased during AVP and remained higher post-AVP also. Similarly, Alpha, Beta and Gamma wave power increased significantly during AVP as compared to pre-AVP and post-AVP. The lungs-heart-brain act as coupled oscillators, the analysed data show an increased arousal, attentive and focused state with a negligible change in the heart rate. An overall increased variability in HRV was recorded.

Health Promoting Lifestyle Profile Assessment among Medical and Nursing Students of Central India.

The Health Promoting Lifestyle Profile-II (HPLP-II) is a western-developed questionnaire that assesses 52 health-promoting behaviours (HPB) over six subscales. The purpose of this research was to identify and assess all six components of the Health-Promoting lifestyle Profile among medical and nursing students in central India (AIIMS and Gandhi Medical College, Bhopal). A cross sectional descriptive study was conducted on 619 medical (MBBS/MD/MS) and nursing students of AIIMS and GMC, Bhopal. The online questionnaires were provided to them which consisted of questions from the Health-Promoting Lifestyle Profile II. Health-promoting behaviors differed by gender in MBBS students particularly with respect to Health-Promoting Lifestyle (p=0.009), Health Responsibility (p=0.021), Nutrition (p=0.046) and Physical Activity (p=0.004). Males had significantly higher scores then females. But the difference between males and females was not significant in PG (MD and MS) students. Scores of five out of six scales (except spiritual growth) were found to be higher among nursing students in comparison with medical students (MBBS, MD and MS). These results will raise concerns about lifestyle behaviours and encourage medical and nursing students to evaluate their own health-promoting lifestyles. Physical exercise and diet should be prioritised, particularly for medical students.

A Study on Psychosomatic Problems Related to the Problematic Internet Use among Adolescents at Selected Schools of Aspur Block, Dungarpur, Rajasthan.

Adolescents' scholastic and social life has become increasingly reliant on the internet and mobile technology in recent years. Problematic Internet Use (PIU) is a growing problem in Indian adolescents. However, in many cases, this use has become obsessive, causing them to neglect other elements of their lives. Adolescents account for over 35% of internet users in India. The purpose of this study was to evaluate the psychosomatic problems related to internet overuse among school going adolescent students. This cross-sectional survey was conducted among 348 students in selected schools of Aspur block in Dungarpur-district, Rajasthan, India in the month of February 2016. Problematic internet use (PIU) was assessed by Young's twenty question internet addiction test. The Depression, Anxiety, Stress Scale (DASS-42) and self structured questionnaire check list was used to measure psychosomatic problems among adolescents. Approximately 5.17% adolescents showed Problematic internet use (PIU). Adolescents with PIU were associated with age between 14-16 year, males,10th class of study, using internet since more than 3 years, being at home, mobile as a source of internet use, purpose of internet use with online gaming. Compared with normal internet users, there were significant correlation between Problematic Internet Use and Somatic Complaints r=0.3660, p<0.05, Depression r=0.3982, p<0.05. Anxiety r=0.2512, p<0.05 and Stress r=0.2886, p<0.05. Problematic internet use (PIU) is an increasing problem among teenagers and it has been linked to psychosomatic issues. Timely Effective measures are needed to prevent the spread of this problem.

Identification of haptoglobin and apolipoprotein A-I as biomarkers for high altitude pulmonary edema.

We have investigated the plasma proteome using 2D gel electrophoresis and matrix-assisted laser desorption/ionization tandem time of flight from patients with high altitude pulmonary edema (HAPE). A complete proteomic analysis was performed on 20 patients with HAPE and ten healthy sea level controls. In total, we have identified 25 protein spots in human plasma and found that 14 of them showed altered changes in HAPE patients, which mainly were acute phase proteins (APPs), compliment components, and apolipoproteins among others. Among the APPs, haptoglobin α2 chain, haptoglobin ß chain, transthyretin, and plasma retinol binding precursor showed overexpression in HAPE patients as compared to controls. To validate the result of proteomic analysis, two proteins were selected for enzyme-linked immunosorbent assay and Western blotting analysis. Our data conclusively shows that two proteins, haptoglobin and apolipoprotein A-I are upregulated in plasma of HAPE patients. These proteins may provide a fast and effective control of inflammatory damage until the subsequent mechanisms can begin to operate. Taken together, our findings further support the hypothesis that inflammatory response system is linked to the pathophysiology of HAPE.

Membrane fusion in organelle biogenesis.

Intracellular membrane fusion is a fascinating reaction that is crucial for cellular function. Several components of the membrane fusion machinery have been identified, although a precise understanding of the fusion mechanism is lacking. More recent studies are revealing novel proteins that regulate membrane fusion during organelle assembly.

The organisation of the Golgi apparatus.

The past year has seen considerable progress in understanding the mechanism of COPI (coatomer protein I) vesicle docking and SNARE (soluble NSF attachment protein receptor) mediated fusion, the mechanism of cisternal growth and stacking and the regulation of Golgi architecture. The route taken by cargo proteins through the Golgi apparatus is still a matter of some dispute.

Acute mixed cellular and humoral rejection of renal allograft with leucopenia.

Diagnosis and management of acute renal allograft dysfunction often pose challenge to nephrologists during practice. Acute rejection is a major cause of acute graft dysfunction but is rare in patients with leucopenia. Acute rejection can have either humoral or cellular components or sometimes mixed components. Mixed acute cellular and humoral rejection often present as steroid resistant rejection. Here we report a patient with live related renal transplant recipient with acute graft dysfunction with leucopenia who was found to have mixed acute cellular and humoral rejection.

Acute Effects on Heart Rate Variability during Slow Deep Breathing.

Slow deep breathing is known to modulate cardiovascular control and is a used in many ancient breath control practices like pranayama. The changes in Heart Rate Variability (HRV) during a less known form of Slow Deep Breathing (SDB) with equal counts of inhalation, holding and exhalation are not well documented. This study analyses the changes during such a slow deep breathing pattern with less than 4 breaths per minute on HRV. The cross sectional descriptive type of observational study was done at AIIMS, Bhopal on 30 regular Kriya yogi volunteers who are practicing for last 10-20 years. SDB involves slow and deep inhalation through the nose, usually to a count of 15, holding for an equal count of 15, followed by slow and complete exhalation for a similar count of 15. The process was repeated for five minutes. The recording ECG for HRV analysis was taken by heart rate variability (Dinamika HRV - Advanced Heart Rate Variability Test System, Moscow, Russia). The resting and during readings of heart rate variability parameters were compared and analysed using a paired t-test. Time domain parameters: Standard Deviation Normal to Normal (SDNN) and Root Mean Square of Successive Differences (RMSSD) were increased at a high level of statistical significance during the manoeuvre. Frequency Domain parameters: Low Frequency (LF), High Frequency (HF), LF/HF ratio increased significantly. Parasympathetic activity is represented by LF when respiration rate is lower than 7 breaths per minute or during taking a deep breath. Thus, when the subject is in the state of relaxation with a slow and even breathing, the LF values can be very high indicating an increase in parasympathetic activity rather than an increase in sympathetic regulation. Practice of pranayama naturally slows the breathing, which in turn makes the heart calmer and calmer as demonstrated by a statistically significant decrease in heart rate after five minutes of SDB. Transient and rapid excitation of cardiovascular system during SDB suggests that the multiple regulatory mechanism at play like mechanical coupling, baroreflex and central mechanisms. Slow yogic breathing (pranayama) may serve as a physiologic method to draw upon cardio-vagal reserve and regular practice of these manoeuvres may beneficially affect cardiovascular autonomic regulation in health and in various cardiovascular diseases.

Effect of Slow and Deep Breathing on Brain Waves in Regular Yoga Practitioners.

Various yogic practices focus on breathing. Breathing may affect brain waves. This cross-sectional study was conducted to see the effect slow and deep respiration on EEG activity in experienced Yoga practitioners at the Department of Physiology at AIIMS, Bhopal, India. The duration of the study was about one year. EEG was taken and was analyzed by Dinamika - Advanced Test System, Moscow, Russia. Paired t-test using graph pad software was used for statistical analysis. The Delta percentage decreased and the other wave's percentages theta, alpha, and beta increased significantly. The person becomes deeply relaxed and more focussed on slow and deep breathing.

Microtubule independent vesiculation of Golgi membranes and the reassembly of vesicles into Golgi stacks.

We have recently shown that ilimaquinone (IQ) causes the breakdown of Golgi membranes into small vesicles (VGMs for vesiculated Golgi membranes) and inhibits vesicular protein transport between successive Golgi cisternae (Takizawa et al., 1993). While other intracellular organelles, intermediate filaments, and actin filaments are not affected, we have found that cytoplasmic microtubules are depolymerized by IQ treatment of NRK cells. We provide evidence that IQ breaks down Golgi membranes regardless of the state of cytoplasmic microtubules. This is evident from our findings that Golgi membranes break down with IQ treatment in the presence of taxol stabilized microtubules. Moreover, in cells where the microtubules are first depolymerized by microtubule disrupting agents which cause the Golgi stacks to separate from one another and scatter throughout the cytoplasm, treatment with IQ causes further breakdown of these Golgi stacks into VGMs. Thus, IQ breaks down Golgi membranes independently of its effect on cytoplasmic microtubules. Upon removal of IQ from NRK cells, both microtubules and Golgi membranes reassemble. The reassembly of Golgi membranes, however, takes place in two sequential steps: the first is a microtubule independent process in which the VGMs fuse together to form stacks of Golgi cisternae. This step is followed by a microtubule-dependent process by which the Golgi stacks are carried to their perinuclear location in the cell. In addition, we have found that IQ has no effect on the structural organization of Golgi membranes at 16 degrees C. However, VGMs generated by IQ are capable of fusing and assembling into stacks of Golgi cisternae at 16 degrees C. This is in contrast to the cells recovering from BFA treatment where, after removal of BFA at 16 degrees C, resident Golgi enzymes fail to exit the ER, a process presumed to require the formation of vesicles. We propose that at 16 degrees C there may be general inhibition in the process of vesicle formation, whereas the process of vesicle fusion is not affected.

A specific activation of the mitogen-activated protein kinase kinase 1 (MEK1) is required for Golgi fragmentation during mitosis.

Incubation of permeabilized cells with mitotic extracts results in extensive fragmentation of the pericentriolarly organized stacks of cisternae. The fragmented Golgi membranes are subsequently dispersed from the pericentriolar region. We have shown previously that this process requires the cytosolic protein mitogen-activated protein kinase kinase 1 (MEK1). Extracellular signal-regulated kinase (ERK) 1 and ERK2, the known downstream targets of MEK1, are not required for this fragmentation (Acharya et al. 1998). We now provide evidence that MEK1 is specifically phosphorylated during mitosis. The mitotically phosphorylated MEK1, upon partial proteolysis with trypsin, generates a different peptide population compared with interphase MEK1. MEK1 cleaved with the lethal factor of the anthrax toxin can still be activated by its upstream mitotic kinases, and this form is fully active in the Golgi fragmentation process. We believe that the mitotic phosphorylation induces a change in the conformation of MEK1 and that this form of MEK1 recognizes Golgi membranes as a target compartment. Immunoelectron microscopy analysis reveals that treatment of permeabilized normal rat kidney (NRK) cells with mitotic extracts, treated with or without lethal factor, converts stacks of pericentriolar Golgi membranes into smaller fragments composed predominantly of tubuloreticular elements. These fragments are similar in distribution, morphology, and size to the fragments observed in the prometaphase/metaphase stage of the cell cycle in vivo.

Reconstitution of vesiculated Golgi membranes into stacks of cisternae: requirement of NSF in stack formation.

We have developed an in vitro system to study the biochemical events in the fusion of ilimaquinone (IQ) induced vesiculated Golgi membranes (VGMs) into stacks of cisternae. The Golgi complex in intact normal rat kidney cells (NRK) is vesiculated by treatment with IQ. The cells are washed to remove the drug and then permeabilized by a rapid freeze-thaw procedure. VGMs of 60 nm average diameter assemble into stacks of Golgi cisternae by a process that is temperature dependent, requires ATP and a high speed supernatant from cell extract (cytosol), as revealed by immunofluorescence and electron microscopy. The newly assembled stacks are functionally active in vesicular protein transport and contain processing enzymes that carry out Golgi specific modifications of glycoproteins. The fusion of VGMs requires NSF, a protein known to promote fusion of transport vesicles with the target membrane in the exocytic and endocytic pathways. Immunoelectron microscopy using Golgi specific anti-mannosidase II antibody reveals that VGMs undergo sequential changes in their morphology, whereby they first fuse to form larger vesicles of 200-300-nm average diameter which subsequently extend into tubular elements and finally assemble into stacks of cisternae.